Method of and device for packing a chromatography column

ABSTRACT

A method and device for packing a chromatography column formed of one or more vibration devices attached to top and/or bottom flanges of the column. Media is added in one or more steps to the column, allowed to settle under the effects of gravity and then subjected to one or more treatments of vibration from the vibration devices until a suitably packed column is obtained. Liquid used to suspend the media while being placed into the column may be at least partially removed before or during the vibration step(s). The remaining liquid is then removed or replaced after the packing has been obtained.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a Continuation Application of U.S.application Ser. No. 12/287,189, filed on Oct. 7, 2008, now U.S. Pat.No. 9,116,150, which claims the benefit of priority of U.S. ProvisionalPatent Application No. 61/003,631, filed on Nov. 19, 2007, the entirecontents of which are incorporated by reference herein.

The present invention relates to a method and device for packing achromatography column. More particularly, it relates to a method anddevice for packing a chromatography column using vibration.

BACKGROUND

Packing chromatography columns is as much an art as it is a science.Even the smallest of columns used in the analytical laboratory can bepacked incorrectly leading to inconsistent results.

The most common problems in packing lead to channeling or gaps in themedia bed through which the liquid and components to be chromatographedpreferentially flow, leading to inconsistent results.

Numerous methods have been developed to attempt to obtain consistentpackings. Many have used a dynamic flow of the media in a slurry formedof the media and a liquid to obtain a suitable packing.

Some have added the use of vibration during this dynamic packing to helpthe consistency of the packing effort. Others have used vibration in theslurry before it is added to the column to create as uniform asuspension of media in the slurry as possible.

While all of these efforts work to a large extent, they all require mucheffort and the use of large volumes of liquid to do so. What is neededis a simple method and device for packing columns regardless of size.The present invention provides such a method and device.

SUMMARY OF THE INVENTION

The present invention is a method and device for packing achromatography column with a chromatographic media. The inventionconsists of a column having one or more vibrational device attached toits outer surface. A slurry of media and a liquid of choice (aqueous ornon-aqueous depending upon the media selected) is put into the column inone or more portions and allowed to settle into a loose bed under theeffect of gravity. The settled bed is then subjected to one or moresequences of vibration from the one or more vibration devices on thecolumn to cause the bed to condense and form a uniformly packed bed. Thecolumn is then closed and the column is run as is typical in the art.

It is an object of the present invention to provide a method of packinga chromatography column comprising the steps of providing achromatography column and a media for packing within the column, forminga slurry with the media and a liquid, adding at least a portion of theslurry to the column, allowing the at least a portion of the slurry tosettle under gravity, vibrating the slurry in the column and removingthe liquid from the column.

It is an object of the present invention to provide a method of packinga chromatography column comprising the steps of providing achromatography column having one or more vibration devices attached toits outer surface and a media for packing within the column, forming aslurry with the media and a liquid, adding at least a portion of theslurry to the column, allowing the media to settle under gravity to forma loose bed, activating the one or more vibration devices to vibratingthe slurry in the column and removing or replacing the liquid from thecolumn to form a packed bed of media.

It is an object of the present invention to provide a method of packinga chromatography column comprising the steps of providing achromatography column having one or more vibration devices attached toits outer surface and a media for packing within the column, forming aslurry with the media and a liquid, adding a first portion of the slurryto the column, allowing the media of the first portion to settle,activating the one or more vibration devices to vibrating the firstportion of slurry in the column while removing the liquid from thecolumn to form a packed bed of media, adding at least a second portionof the slurry, allowing at least the second portion of the media tosettle under gravity, activating the one or more vibration devices tovibrating the second portion of slurry in the column and removing orreplacing the liquid of the slurry from the column to form a packed bedof media.

It is an object of the present invention to add the slurry in two ormore steps and the slurry is vibrated after each addition of slurry.

It is an object of the present invention to provide a hard media for themethod selected from porous glass, porous silica and porous plastics.

It is an object of the present invention to provide two or morevibration devices evenly spaced from each other around a circumferenceof the column wall.

It is an object of the present invention to provide a device for packinga chromatography column comprising a column having a vertical wall, thevertical wall having an inner and outer surface, a lower plate havingone or more ports, said lower plate being selected from the groupconsisting of fixed plates and adjustable plates, an adjustable upperplate and one or more vibration devices mounted to the column.Preferably, the one or more vibrator devices are located either on theflange of the lower plate or the top flange of the column wall.

It is an object of the present invention to provide two or morevibration devices are evenly spaced apart from each other around acircumference of the outer surface of the vertical wall.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a device according to the present invention in crosssectional view.

FIG. 2 shows a bar graph with the HETP results of Example 1.

FIG. 3 shows a bar graph with the HETP results of Example 2.

FIG. 4 shows a bar graph with the results of Example 2 over time.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to a method and device for packing achromatography column.

FIG. 1 shows one device that can be used in the present invention. Itcomprises a column 2 having a vertical wall 4 that forms the body of thecolumn. The vertical wall 4 has an inner surface 5 and an outer surface7. The area 9 within the inner surface 7 of the wall 4 provides thespace 9 for the chromatography media and the lower plate 6 and upperplate 10.

The lower plate 6 is attached to the bottom of the vertical wall as iswell known in the art. The lower plate 6 may be fixed or adjustable(movable) into or out of the column as is desired. A port 8 is formed inthe lower plate 6 and is in selected fluid communication with the space9 generally through a valve arrangement (not shown) that selectivelyopens and closes access to the space 9 as desired.

The upper plate 10 or adjuster plate as it is commonly called isattached above the wall 4 to the column 2 and is movable above andwithin the space 9 to provide a selectively closed environment for themedia (not shown) in the space 9. One or more ports 11 are formed in theupper plate and are in selected fluid communication with the space 9generally through a valve arrangement (not shown) that selectively opensand closes access to the space 9 as desired.

The use of threaded rods 12 are used to support and connect the upperflange 13 to the bottom flange 14 with column wall 4 in between in aliquid tight manner. Screw 15 mounted through the upper flange 13 andattached to the upper plate 10 allows the upper plate to move into andout of the space 9 as needed. While shown as being manually driven byturning device 17 it can be automatically driven and can use hydrauliclifts (not shown) if desired to pull the upper plate 10 out of and abovethe wall 4 to expose the space 9 for loading of media or repairs.

One or more vibration devices 16 are placed adjacent to the outersurface 7 of the wall 4. Preferably, they are temporarily attached toeither the upper or lower flange 13, 14 or both so they may be removedduring cleaning or chromatographic operations to avoid damage to thedevices 16. If more than one device 16 is used, it is preferred thatthey be evenly spaced from each other about the circumference of thewall 4. As shown in FIG. 1, at least one device 16 is mounted to thelower flange 14 through the use of two or more clamps 18, such asC-clamps.

Vibration devices can be selected from a range of pneumatically drivenroller or turbine vibrators. These vibrators are capable of generatingvibrations with frequencies in the 6000-30000 Hz. The frequency ofvibrations can be controlled by controlling the supply pressure. Suchdevices are available from OLI Vibrators, Inc and are commonly used inthe powder processing Industries.

A method of using the device is as follows.

A column having one or more vibration devices arranged on the column,preferably on its top and/or bottom flange.

A chromatography media is formed into a slurry with a liquid which canbe aqueous based such as water, saline solutions, various chromatographybuffers, water alcohol blends and the like or non-aqueous based such asvarious alcohols depending on the media selected. The amount of liquidused should be sufficient to create a free flowing slurry. Typically theliquid will be from about 10 to about 90% of the volume of the slurry,preferably 30-70% and more preferably about 30 to about 50% of thevolume of the slurry.

The slurry is added to the column either through the open top of thecolumn into the space 9 as the upper plate 10 has been moved out of thecolumn or through the one or more ports 11 formed in the upper plate 10or through an ISOPAK® valve (EMD Millipore Corporation of Billerica,Mass.). The media is allowed to settle out of the slurry.

After settling under the effect of gravity, the media is in the form ofa loose bed. One or more sequences of vibration are applied to the bedthrough the one or more vibration devices 16 arranged on the columnbottom or top flange. This is preferably followed by flow of liquidthrough the column to create a compact uniform bed.

In another embodiment of the present invention, settling can beconducted with periodic intervals of vibration such that a period ofvibration is followed by a period of settling without vibration untilthe bed of desired packing consistency is obtained.

In a further embodiment, the use of the flow of fluid through the mediaas it settles can in some instances help with the settling and packing.This maybe a steady flow or intermittent or pulsed flow cycles of one ormore times. Intermittent flows can be from about 30 seconds to about 5minutes each and when used more than once in a single packing step canbe spaced apart as needed, preferably from about 1 minute to about 10minutes between each flow pulse.

In a different embodiment the combination of flow packing and vibrationduring the bed formation can be used.

The amount of time for vibration and when it is used will vary dependingupon the media selected, the liquid used in the slurry, the depth of thebed desired and the desired level of packing to be obtained and can beeasily determined with simple experimentation by one of ordinary skillin the art. Typically, the use of one or more vibration sequences offrom about 1 to about 10 minutes, preferably from about 1 to about 5minutes can be used.

In those embodiments in which vibration occurs at least during settling,it is preferred that the vibration be for about 30 seconds to about 3minutes followed by no vibration for a period of from about 1 minute toabout 5 minutes, preferably the sequence is for about 1 minute ofvibration followed by about 2 minutes of no vibration. This can berepeated if necessary or desired as many times as needed to obtain thedesired packed bed.

In either embodiment, the upper plate 10 is then moved against the uppersurface of the bed and the chromatography bed is now ready forequilibration and/or use.

This invention works with all types of chromatography media includingsofter or compressible media such as agarose based media such asSepharose® media or MabSelect® media available from GE Healthcare orhard media such as porous silica, porous plastic such as POROS® mediafrom Perseptive Biosystems Inc, or porous glass, preferably controlledpore glass such as CPG® media from EMD Millipore Corporation, moreparticularly controlled pore glass that has a ligand attached to it,such as ProSep® A media from EMD Millipore Corporation.

EXAMPLES Example #1

A QuikScale® column (EMD Millipore Corporation of Billerica, Mass.) of450 mm diameter was obtained. A known quantity of CPG® controlled poreglass chromatography resin of 100 μm diameter and a pore size of 1000 A(EMD Millipore Corporation of Billerica, Mass.) was transferred to thecolumn in a slurry formed of the resin and reverse osmosis (RO) water.This quantity of resin was packed in four different ways:

-   -   1. Gravity—Allow the packed bed to form by settling under        gravity alone    -   2. Flow pack—Allow the packed bed to form by application of flow    -   3. Stop Flow—Form the packed bed by flow pack and then subject        it to 4-5 cycles of periodic flow (flow on for 2 minutes and off        for 3 minutes)    -   4. Vibration—Allow the packed bed to form by allowing to settle        under gravity with periodic input of vibrational energy (on for        1 minute and off for 2 minutes). The vibrational energy was        input to the column by clamping a single OR65 vibrational device        (OLI Vibrators, Inc, Norcross, Ga., USA,) on the bottom flange        of the column.

The resin was unpacked and re-slurried prior to each packing method.Each packed bed was qualified using an injection of a 0.5M Sodiumchloride solution in RO water. The Height Equivalent to a TheoreticalPlate (HETP) of the sodium chloride peak exiting the packed bed was usedas the comparison metric. The lower the HETP, the more efficient was theresultant bed. The HETP obtained with each packed bed is illustrated inFIG. 2. As is evident from the figure, the vibration packing techniqueyielded the most efficient bed.

Example #2

An IPP 1600×600×500 column rated to 3 bar (EMD Millipore Corporation ofBillerica, Mass.) of 1.6 meters (hydraulically operated and fitted withIsoPak® valves in both plates) was obtained.

A known quantity of CPG® controlled pore glass chromatography resin of100 micron diameter and a pore size of 1000 angstroms (EMD MilliporeCorporation of Billerica, Mass.) was transferred to the column in aslurry formed of the resin and reverse osmosis (RO) water. The media waspacked the following ways:

-   -   1. Stop flow with fluidization—Following transfer of media into        the column, the media was allowed to settle under gravity,        subsequently it was fluidized at 100 cm/hr for 2 column volumes        and finally flow was applied to form a settled bed. Following        the formation of the settled bed, the latter was subjected to        4-5 cycles of periodic flow (flow on for 2 minutes and off for 3        minutes)    -   2. Stop flow with vibration—Following transfer of media into the        column, flow was immediately applied to the column to form        settled bed. Following the formation of the settled bed, the        latter was subjected to periodic vibration followed by flow (1        min vibration+1 minute flow). The vibrational energy was input        to the column by clamping a single OR100 vibrational device (OLI        Vibrators, Inc, Norcross, Ga., USA,) on the bottom flange of the        column.    -   3. Vibration (1 vibrator)—Following transfer of media into the        column, the media was allowed to settle under gravity with        periodic input of vibrational energy (1 minute on, 2 minutes        off) till the formation of a stable packed bed. The vibrational        energy was input to the column by clamping a single OR100        vibrational device (OLI Vibrators, Inc, Norcross, Ga., USA,) on        the bottom flange of the column.    -   4. Vibration (3 Vibrators)—Following transfer of media into the        column, the media was allowed to settle under gravity with        periodic input of vibrational energy (1 minute on, 2 minutes        off) till the formation of a stable packed bed. The vibrational        energy was input to the column by clamping three OR100        vibrational device (OLI Vibrators, Inc, Norcross, Ga., USA,) on        the bottom flange of the column and evenly spaced from each        other.

Each packed bed was qualified using an injection of a 0.5M Sodiumchloride solution in RO water. The Height Equivalent to a TheoreticalPlate (HETP) of the sodium chloride peak exiting the packed bed was usedas the comparison metric. The lower the HETP, the more efficient theresultant bed.

The HETP obtained with each packed bed is illustrated in FIG. 3. As isevident from the figure, inclusion of vibration in the packing techniqueimproves the performance of the bed. The use of vibration alone, whenthe vibrational energy is input with three evenly spaced vibratingdevices yields the most efficient bed.

The packed bed obtained by vibrating with three vibrational devices wassubjected to continuous flow for close to 500 column volumes (CV) totest for its stability. FIG. 4 illustrates the HETP of qualificationsodium chloride (0.5M in RO water) pulses at the beginning and end ofthe stability test. As is evidenced by the lower HETP at the end of thetest, the packed bed was stable to continuous operation.

Thus, Examples 1 and 2 demonstrate that the use of vibration, inconjunction with flow or without flow, yields more efficient and stablepacked beds than with flow alone.

What is claimed:
 1. A method of packing a chromatography columncomprising the steps of: a. providing a chromatography column having oneor more vibration devices attached to its outer surface; b. forming aslurry with a media and a buffer solution; c. transferring the slurryinto the column and immediately applying flow for 1 minute to settle theslurry in the column; d. activating the one or more vibration devices togenerate vibrations for 1 minute following the settling of the slurryunder flow for 1 minute; e. repeating flow for 1 minute followed byvibrations for 1 minute, until a packed bed of media is obtained.
 2. Themethod of claim 1, wherein the media comprises controlled pore glasswith a Protein A ligand.
 3. The method of claim 1, wherein the buffersolution is present from about 10 to about 90% of the volume of theslurry.
 4. The method of claim 2, wherein the controlled pore glassmedia has a 100 μm diameter and pore size of 1000 A.
 5. The method ofclaim 1, wherein the column comprises two or more vibration devices onthe outer surface of the column.
 6. The method of claim 5, wherein thetwo or more vibration devices are evenly spaced apart on the outersurface of the column.
 7. The method of claim 1, wherein the columncomprises three or more vibration devices on the outer surface of thecolumn.
 8. The method of claim 7, wherein the three or more vibrationdevices are evenly spaced apart on the outer surface of column.
 9. Themethod of claim 1, wherein the one or more vibration devices are clampedonto the outer surface of column.
 10. The method of claim 1, wherein thecolumn comprises a bottom flange and the vibration devices are evenlyspaced on bottom flange of the column.
 11. The method of claim 1,wherein the packed bed of media has a lower Height Equivalent to aTheoretical Plate (HETP) compared to a column packed using flow andfluidization.